Process for producing n-formyl hydroxyamino acetic acid



United States Patent 3,254,001 PROCESS FOR PRODUCING N-FORMYLHYDROXYAMINO ACETIC ACID Edward A. Kaczka, Union, and Eugene I...Dulaney, Summit, N.J., assignors to Merck 8; (10., Inc., Railway, N .J acorporation of New Jersey N 0 Drawing. Original application Apr. 29,1960, Ser. No. 25,506, now Patent No. 3,202,700. Divided and thisapplication Sept. 16, 1964, Ser. No. 397,000

14 Claims. (Cl. 195-29) This application is a division of copendingapplication Serial No. 25,506, filed April 29, 1960, now US. Patent Alsoincluded within the invention are the salts and esters of 'N-formylhydroxyamino acetic acid, so that the generic structural formula may bepictured as:

O 0HCN0H2(i0R where R may be hydrogen or a metal, and R may be hydrogen,a metal, or a hydrocarbon group.

In addition to the free acids (where R and R are hydrogen), the alkalimetal and alkaline earth metal salts, such as the sodium, potassium,lithium, calcium, and barium salts, and the lower alkyl estersexemplified by methyl, ethyl, propyl, and isopropyl esters, representthe preferred embodiments of R and 12,. However, heavy metal salts andother esters such as the benzyl, phenethyl, allyl, and like esters maybe produced according to the invention.

As will be discussed more fully below, the compounds of this inventionare effective in inhibiting or preventing the growth of certainundesirable plants such as crab grass, dandelions, poison ivy, and otherobnoxious weeds, and thus represent important and highly usefulcontributions to the agricultural field.

In accordance with the invention, it has been discovered that N-formylhydroxyamino acetic acid may be produced in good yields by fermentationof a nutrient medium with a suitable microorganism. The organismemployed for this purpose is a strain of Penicillium of theMonov'erticallata or Asymmetrica conidial structure type, and preferablyone classified as Within the Penicillium frequentans, Penicilliumlividum, Penicillium janthinellum, Penicil- Izum thomii, Penicilliumimplicatum, Penicillium camemberti or Penicillium expansum series. For adiagrammatic key to the classification of Penicillia reference is madeto the text Manual of the Penicillia, by Raper and Thom, The Williams &Wilkins 00., 1949, pages 120421. Microorganisms which are particularlysatisfactory for production of 'N-formyl hydroxyamino acetic acid arestrains of the Penicillium frequentans and Penicillium pwrpurrescensspecies of the Penicillium frequentans series, and of the Penicilliumaurantio-violaceum species of the Penicillium lividum series.

In order to prepare N-forrnyl hydroxyamino acetic acid by thisfermentation method, a producing strain of Penicillium of the typedescribed above is grown under submerged aerated conditions in anutrient medium containice tract, soybean meal, cotton seed meal,casein, distillers solubles and the like. The fermentation medium alsocontains trace amounts of salts which are normally contained in thecarbon and nitrogen sources. The nutrient medium is normally adjusted toa pH in the range of 6-8, and sterilized before inoculation with theproducing strain of Penicillium. Substantial quantities of N-formylhydroxyamino acetic acid are normally produced in the fermentationmedium after a growth period of approximately 4-10 days at temperaturesin the range of 25- 30 C. With the preferred microorganism, about 2-15grams of N-forrnyl hydroxyamino acetic acid per liter of fermentationbroth is produced under these conditions.

In order to recover the N-fonmyl hydroxyamino acetic acid from thefermentation broth, the mycelia are first removed by techniques such asfiltration or centrifugation. The filtered broth is concentratedessentially to dryness by techniques known in this art. The solidresidue is then extracted with methanol and the methanol extracts, whichcontain .the desired product, are concentrated to dryness or neardryness. Final purification is achieved by crystallization from aqueousethanol. This method of recovering N-formyl hydroxyamino acetic acid maybe employed to obtain either the free acid or an alkali metal saltthereof. Unless pH adjustments are made at conclusion of fermentation orduring the isolation process, the product obtained will depend upon thepH of the finished fermentation broth. It is normally preferred to startthe fermentation at a pH of 6-8.

If it is desired, Nsformyl hydroxyamino acetic acid may be synthesizedby bringing the producing enzyme system elaborated by theabove-described microorganisms into intimate contact with an aqueousmedium containing sources of carbon and nitrogen. With this procedure,it is not necessary or contemplated that a growing culture of thePenicillium be used.

N-formyl hydroxyamino acetic acid, and its salts and esters, exhibitabsorption in the infra-red portion of the spectrum, give a red-purplecolor with ferric chloride, do not reduce Fehlings solution, decolorizepermanganate, give a yellow colow with bromphenol blue and an atypical'ninhydrin test. They may be hydrolyzed with acid to hydroxyamino aceticacid and formic acid.

It has been found convenient in the case of the N- formyl hydroxyaminoacetic acid prepared by fermentation, to recover the product as analkali metal salt such as the sodium salt. The salts have the activityof the parent acid and may be employed as such or converted to the acidby treatment with an aqueous mineral acid or by ion exchange resinmetathesis. N-formyl hydroxamino acetic acid exists in two crystallineforms, cubes melting at 108110 C. and needles melting at 119l20 C.

Salts of N-formyl hydroxyamino acetic acid are also readily obtained bytreating a solution of acid with an appropriate base such as ammoniumhydroxide, potassium hydroxide, sodium carbonate, calcium carbonate,lithium hydroxide, and the like. It will be realized that monoanddi-salts can be produced depending on the amount of base employed, thedi-salts forming at a pH of about 10 and above.

Similarly, esters are synthesized by treatment of the N-formyl acid witha suitable esterfying agent such as a diazoalkane. In this manner, loweralkyl esters such as the methyl, ethyl, and propyl esters of N-formylhydroxyamino acetic acid are obtained, which compounds possess theactivity of the acid and salts discussed in more detail hereinbelow.Alternatively, esters may be obtained by transesterification of themethyl ester with an appropriate alcohol, or by esterification ofhydroxyamino acetic acid and subsequent N-formylation of the ester. Thislatter method is convenient since the esterification may be acidcatalyzed without risk of hydrolyzing the N-formyl group.

The monosodium salt of N-formyl hydroxyamino acetic acid, which is thespecies normally recovered from a fermentation broth, exists as ahydrate as well as in anhydrous form as shown by infra-red spectradeterminations. -The spectra can be divided into two categories: (at)those which show free hydroxyl absorption (band at 2.8;) and b) those inwhich this band is absent. The anhydrous material has characteristicinfra-red absorption at the longer wavelengths which is readilydistinguishable from the hydrate.

The characteristic peaks of absorption in the infra-red region of thespectrum of the two forms of sodium N- formyl hydroxya mino acetate, aswell as the absorption peaks of N-forrnyl hydroxyamino acetic acid, andthe ammonium, potassium, and calcium salts thereof, are at the followingwavelengths in microns:

3.1-5.0 (broad), 5.9-6.1, 6.3, 6.5, 7.7, 8.3, 10.2,

Potassium N-formyl hydroxyamino acetate:

3.0-4.7 (broad), 6.0, 6.5, 7.6, 8.2, 8.4, 9.9', 10.2.

Calcium N-formyl hydroxyarnino acetate:

The compounds of this invention are highly effective in killing orinhibiting the growth of certain types of plants. They are particularlyuseful in that they kill or inhibit growth of weeds and otherundesirable plants such as crab grass, dandelions, sheep sorrel,plantains and wild brush. The treatment is normally carried out byspraying a solution of the N-formyl hydroxyamino acetic acid (or salt orester derivative thereof) on the plants, preferably to run-off.Effective results are obtained with small amounts of active ingredient,i.e. with solutions containing from 30-1000 p.p.m. of N-formylhydroxyamino acetic acid.

In addition to using solutions of pure or substantially pure compoundsfor this purpose, highly satisfactory results are obtained withconcentrates of the. active materials. This is of particularsignificance in the case of N- formyl hydroxyamino acetic acid compoundsbecause it permits the use of crude fermentation concentrates, and evenof the fermentation broth itself, for control of weeds and otherundesirable plants. The N-formyl hydroxyamino acetic acid describedherein likewise inhibits the growth of Alaska pea plants, germinatedtomato seeds and pinto bean plants. This property may be convenien'tlyused as a means of' determining the presence and concentration of ournew compounds in a material,

or as a method of standardizing preparations thereof.

The following examples are given for the purpose of illustration onlyand not by way of limitation.

The spectra were taken on a Perkin-Elmer Model 137 Infra-redSpectrophotometer (Infracord) on a sample suspended ln mineral oil(Nujol).

4 Example 1 A. A nutrient medium having the following composition isprepared:

Distilled water 93.0 400 ml. of this medium is added to a 2 literErlenmeyer bafiled flask and the pH adjusted to 6.8 with 0.1 N sodium Itis then sterilized in an autoclave at 15 pounds per square inch gaugepressure, at.121 C. for 17 The medium is then cooled to roomtemperahydroxide.

minutes. ture and inoculated with 50 ml. of a growing culture ofPenicillium purpurrescens (NRRL N0. 2819). The inoculated medium isincubated on a rotary shaker, operating at a speed of rpm. for 7 days ata temperature of 28 C. At the end of this time, the fermentation brothis filtered through a Biichner filter funnel precoated with Super-Cel.The filtered fermentation broth containing N-formyl hydroxyamino aceticacid inhibits the growth of pinto bean plants, as evidenced by asignificant reduction in width of the trifoliate leaflets of young pintobean plants sprayed with the filtered broth as compared to the width ofthe leaflets of u-nsprayed plants.

B. When a filtered fermentation broth containing N- formyl hydroxyaminoacetic acid and produced in the manner described above is sprayed torun-01f on plots of crab grass, a substantially complete killing of thecrab grass is effected over a period of 28 days after spraying. Whenlawn grasses are similiarly treated, the growth of such grasses issubstantially stopped for a period of about 28 days but grass is notkilled.

C. The inoculum for the fermentation described in Part A above isprepared in the following manner: A culture of Penicillium purpurrescens(NRRL No. 2819) is grown for 7 days at 28 C. on a slant medium composedof 1.0% dextrose, 0.5% hydrolyzed lactalbumin (Edamin), 1.0% yeastextract, 2.0% agar, and the remainder distilled water.

To this slant culture is added 10 m1. of the salt solution medium(described by Davis & Mingioli, J. Bact. 60, 17-28 (1950) without sugar)having the following composition:

Gms./ liter K HPO 7 KH PO 3 NaCitrate-2H O 0.5 MgSO., 7H O 1 (NH SO 1 5ml. of the resulting s'pore suspension is added to 50 ml. of asterilized medium containing 4.0% dextrose, 1.0% cornsteep liquor, 2.0%hydroylzed 'lactalbumin (Edamin), and the remainder distilled water. ThepH of this medium is adjusted to 6.8 with sodium hydroxide prior tosterilization. This medium is incubated in a 250 ml. flask at 28 C. for7 days, the incubation being carried out on a shaker operating at aspeed of 220 rpm. This broth is employed for the inoculation o thefermentation described in Part A above. 1

Example 2 A nutrient medium h aving the following composition:

Percent Dextrose 4.0 C-ornsteep liquor 1.0 Hydrolyzed lactalbumin('Edamin) 2.

Dis-tilled water 93.0

is prepared and 400 ml. thereof added to each of 43 2 liter baffledErlenmeyer flasks. The pH of the medium portions is then adjusted to 6.8with a solution of 0.1 N sodium hydroxide, and each flask sterilized inan autoclave at 15 pounds per square inch gaugepressure at 121 C. lfOI17 minutes. After sterilization, the fiasks are cooled to roomtemperature.

Each of the 43 fiasks are inoculated with 50 ml. of a growing 'cultureof Penicillium frequentans (NRRL No. 2818). The inoculum is prepared inthe same manner described in Example 1(C). The inoculated media areincubated on a rotary shaker, the table of which is operating at 120r.-p.m. tor a period of 7 days at a temperature of 28 C. The resultingbroths are filtered through a Super-Cel coated Biichner filter funneland the filtered fermentation broths are combined.

Three liters of the pooled fermentation broth thus obtained arelyophylized. The total yield of solids from this 3 liter portion is 245g. These solids are triturated with one 1000 ml. and two 700 ml.portions of methanol. The insoluble materiel is separated by filtrationand the methanol solutions combined and evaporated in vacuo to neardryness. This residue thus obtained is divided into two equal portionsand treated as follows:

Portion A is washed with ethanol and the remaining solids dried. Thismaterial, which weighs about 20 g., is dissolved in 100 ml. of Water andthe aqueous solution diluted with 250 ml. of ethanol. Crystallization ofthe sodium salt of N-formyl hydroxyamino acetic acid begins almostimmediately and is essentially complete after one hour. The salt isseparated by filtration and airdried; yield g.

Portion B is dissolved in 100 ml. of water and ethanol added to theaqueous solution until crystallization of sodium N-formyl hydroxyaminoacetate is complete. The yield of the air-dried salt is 10.8 g.Recrystallization of this material from aqueous ethanol givessubstantially pure material, M.P. 205-210 C. (dec.).

Anal-Cale. [for C H NO Na (molecular weight 141.06): C, 25.54; H, 2.86;N, 9.93; Na, 16.3. Found: C, 25.50; H, 2.84; N, 9.91; Na, 16.7.

Example 3 A.- One g. of monosodium N-formyl hydroxyamino acetate isdissolved in 10 of water and the solution passed through a columncontaining ca. 27 ml. of a cation exchange resin of the nuclear sulfonicacid type (Amberlite IR-lZO) on the hydrogen cycle. The resin is washedwith 10 ml. of water and the efiluent and wash combined. The totalvolume is about 20 ml. This is lyophilized and the resulting solidstriturate-d with acetone. The acetone solution is evaporated to drynessin vacuo and the solid thus obtained is triturated with ethanol. Theethanol solution is evaporated to dryness in vacuo. The resultingcrystalline residue consisting ot 'N-formyl hydroxyamino acetic acid isfurther purified by recrystallization trom acetonea'petroleum ether(3060 C.). The crystalline (needle-like) N-formyl hydroxyamino aceticacid obtained melts at 1'19-120 C.

Anal-Cale. rfor C H NO (molecular Weight 119.08): C, 30.26; H, 4.23; N,11.76. Found: C, 30.88; H, 4.48: N, 12.21.

Potentiometric titration in water with 0.1 'N LiOH shows two acidicfunctional groups: pH 1/2 ca. 3.5, eq.

wt. 123, and pH 1/2 ca. 9.1, eq. wt. 11 1.

The acid and its salts have an Rf of 0.3-0.5 in a n butanolzaceticacidcwater (4:121 by volume) system on whatman No. 1 filter paper.

B. 269 mg. of monosodium Naformyl hydroxyamino acetate is dissolved in 2ml. of water and the solution adjusted to ca. pH 1-2 with dilutehydrochloric acid. The solution is evaporated to near dryness in vacuoand the wet residue triturated with three 4 ml. portions of ethanol. Thesolutions are combined and evaporated to dryness in vacuo. The residueis dissolved in '5 rnl..of ethanol and ca. 3 ml. of chloroform added tothe solution. A small amount of white precipitate is separated and theclear solution evaporated to dryness in vacuo. The

6 partially crystalline residue of N-formyl hydroxyamino acetic acid isdissolved in ca. 4 ml. of ethanol and the solution diluted with 35 ml.of chloroform. This solution is evaporated to dryness in vacuo to givecube-like crystals of N-tformyl hydroxyamino acetic acid, M.P. 108 -1il0C. Example 4 Five g. of sodium N-formyl hydroxyamino acetate isdissolved in 20 ml. of water and the resulting solution passed through acolumn containing 200 ml. of Amberlite IR-120 resin on the hydrogencycle. When the sodium salt solution has been put over the resin, theresin is then washed with 50 ml. of water and this wash combined withthe colulmn eflluent. The volume of the resulting solution is about 65ml. This solution is divided into three portions of 21 rnl. each.

One portion is treated with concentrated aqueous ammonium hydroxide to apH of 8 and the basic solution evaporated to dryness under reducedpressure. The residual monoammonium salt of N-formyl hydroxyamino aceticacid is purified by dissolving in 5 ml. of water and inducingcrystallization by addition of 50 ml. of ethanol to the aqueoussolution.

The second 21 ml. portion obtained above is treated to a pH of 7-8 withdilute aqueous potassium hydroxide. This solution is then concentratedto dryness and the residue dissolved in 5 ml. of water. Addition of 35ml. of ethanol to the aqueous solution results in crystallization of themonopotassium salt of N-formyl hydroxyamino acetic acid.

The third portion is treated with excess calcium carbonate. Afterevolution of carbon dioxide is complete, the excess solid calciumcarbonate is removed by centrifugation and the clear solution thusobtained concentrated to near dryness. The crystalline material whichforms is the calcium salt of N-formyl hydroxyamino acetic acid which isonly slightly soluble inwater.

Example 5 0.5 mg. of N-formyl hydroxyamino acetic acid is dissolved inabout 0.5 ml. of dilute aqueous sodium hydroxide. Dilution of theresulting solution with ethanol causes crystallization of the di-sodiumsolution. Disodium N-formyl hydroxyamino acetate may also beobtainedfrom the monosodium salt by adjusting the pH of a solution containingthe monosalt to about 11-12 with dilute sodium hydroxide. Upon additionof ethanol to this strongly basic solution, the di-sodium saltcrystallizes and may be purified by recrystallization from aqueousethanol.

. Potentiometric titration of the di-sodium salt with 0.1 N hydrochloricacid gives two spans-pH /2 ca. 8.7 and pH /2 ca. 2.9.

The di-sodium salt of N-formyl hydroxyaminoacetic acid obtained asdescribed above may be converted, if

desired, to the monosodium salt by treatment of an aqueous solution to apH of 6-7 with a small amount of glacial acetic acid. The product iscrystallized by dilution of the reaction mixture with ethanol.

Example 6 N-formyl hydroxyamino acetic acid is produced in substantialquantities when the process of Example 2 is carried out employingspecies of Penicillium other than the Penicillium frequentans (NRRL No.2818) employed in that example. Species of Penicillium which are particularly useful in elaborating N-formyl hydroxyamino acetic acid are thefollowing:

Penicillium spinulosum, NRRL 2827, Penicillium spinulosam, NRRL 2829,Penicillium freqaentans, NRRL 1915, of the Penicillium frequemansseries.

Penicillium trzebinskii, NRRL 732, Penicillium aarantio-violaceum, NRRL762, Penicillium livz'a'um, NRRL 754, of the Penicillium lividum series.

Penicillium implicatum, NRRL implicatum series.

Penicillium turbatum, NRRL 758, of the Penicillium thomii series.

Penicillium janthinellum, NRRL 2828, of the Penicillium janlhinellumseries.

Penicillium caseicolum, NRRL 876, of the Penicillium camemberti series.

Penicillium crustosum, NRRL 967, of the Penicillium expansum series.

723, of the Penicillr'um Example 7 To an ethereal solution of about 2.5g. of N-forrnyl hydroxyamino acetic acid, an ethereal solution of 1 g.of diazomethane is added dropwise with stirring. The reaction is carriedout at room temperature. When addition is complete, the ether is removedin vacuo and the residue dissolved in acetone. Crystalline methylN-formyl hydroxyamino acetate is obtained by diluting the solution withpetroleum ether (3060 C.). The ester melts at 7172 C.

The infra-red absorption spectrum of a substantially pure sample ofmethyl N-formyl hydroxyamino acetate suspended in mineral oil (Nujol)taken on a Perkin- Elmer Model 137 Infrared Spectrophotometer(Infracord) shows characteristic peaks at the following wavelengths,expressed in microns: 5.75, 6.0, 7.9, 8.25, 8.45, 9.8, 9.95, 10.35,11.15, 12.95, 13.3-13.8 (broad), 14.5.

Example 8 A. The activity of sodium N-formyl hydroxyamino acetate incontrolling crab grass may be demonstrated by spraying a solutionthereof on carb grass grown in greenhouses. In one experiment, potscontaining 30-40 crab Parts per million: Percent kill 4000 99 2000 801000 40 Similar results are obtained when the free acid or other alkaliand alkaline earth metal salts are used instead of the monosodium salt.

B. A fermentation broth containing N-formyl hydroxyamino acetic acid isproduced by fermenting the nutrient medium described in Example 1 abovewith Penicillium purpurrescens NRRL 2819 by the procedure of Example 1.On completion of the fermentation, the solid ma terial is removed byfiltering and the filtered broth sprayed to run-off on two separateplots of crab grass and on an established lawn plot in which there werefive difier ent lawn grasses: Highland Bent, New Zealand fescue, FancyRed Top, Kentucky Blue, and Merion Blue. The following table illustratesthe effects of spraying the strained fermentation broth on the threeplots 28 days after spraying:

Crab grass 2' 6-9 cm. tall 100% killed.

Crab grass: 15-20 cm. tall 95% killed.

Lawn grass Growth stopped; grass not killed.

N-formyl hydroxyamino acetic acid, and its salts and esters, are alsohighly effective in killing sheep sorrel, plantain, dandelions, clover,poison ivy, smart weed and ditch bank weed when aqeuous solutionsthereof are sprayed to run-off on the palnts. Aqueous solutions of thepure substances or of concentrates such as fermentation solids, orfiltered fermentation broths themselves may be employed.

8 Example 9 The following procedure is usedto determine theeffectiveness of the compounds of the invention in inhibiting growth ofpinto bean plants:

Two seeds of pinto 'bean (Phaseolus vulgaris L.) are planted 1 inch deepin a 3 /2 inch water proof paper pot containing a mixture of 50% sandyloam soil and 50% Michigan hyper humus. After the pot is maintained withnormal watering at a temperature of 2838 C. at a 65% relative humidityin the presence of natural sunlight for one week, the less developedplant is removed from the pot and discarded. The soil of the pot is thenfertilized with an aqueous nutrient solution containing calcium nitrate(8.1 g./l.), potassium nitrate (2.5 g./l.), potassium dihydrogenphosphate (0.63 g./l.), and magnesiumsulfate (1.2. g./l.). Three dayslater, when the primary leaves of the remaining plant are abouttwothirds of their full size and when the trifoliate leaves are juststarting to expand, the entire plant, including both sides of theleaves, is sprayed to run-off with the test solution. A second pot,started at the same time and treated the same way, except that the beanplant is not sprayed, is used as a control. The test plant and thecontrol plant are maintained with normal watering at 28 C. and 65relative humidity in the presence of natural sunlight. Four days laterthe width of each leaflet of'the trifoliate leaves of the test plant andof the control plant are measured. Four plants are used for each sampleand four plants for the control.

A. In an experiment using aqueous solutions containing varyingconcentrations of sodium Nformyl hydroxyamino acetic acid the followingresults were obtained:

Parts per million of Width of leaflets, mm.

B. Three liters of whole broth produced by fermentation of a nutrientmedium with Penicillium purpurrescens (NRRL N0. 2819) in a mannersimilar to that described in Example 1 is acidified to a pH of 4 withacetic acid. The acidified broth is filtered and 1600 ml. of the clearfiltrate freeze-dried. Two aqueous solutions containing respectively 10mg./ml. and 20 mg./ml. of the freezedried solids were tested forinhibition of pinto bean plant growth by the above procedure. Thesolution containing 20 mg./ml. of the freeze-dried solids gave a 92%reduction in' width of the trifoliate leaflets as compared with anuntreated control plant, and the solution having 10 mg./ml. of thefreeze-dried solids gave a 69% reduction under the same conditions.

Any departure from the above description which conforms to the presentinvention is intended to be included within the scope of the claims.

What is claimed is:

1. The process for producing N-formyl hydroxyamino acetic acid whichcomprises aerobically cultivating a strain selected from the groupconsisting of Penicillium frequentans, Penicillium purpurrescens,Penicillium aurantia-violaceum, Penicillium trzebinskii, Penicillium,spinulosum, Penicillium lividum, Penicillium janthineluntil asignificant amount of N-formy-l hydroxyamino acetic acid is produced.

'3. The process for preparing an N-formyl hydroxyamino acetate compoundselected from the group consisting of N-formyl hydroxyamino acetic acidand salts thereof which comprises aerobically cultivating an N- formylhydroxyamino acetic acid producing strain of a Penicillium possessing aconidial structure of a .type selected from the group consisting ofMonovertic-illata and Asymmetrica in a culture medium comprisingassimilable sources of carbon and nitrogen and in the presence of aninorganic salt forming cation, until a significant amount of saidN-formyl hydroxyamino acetate compound is obtained in said medium, andrecovering said compound.

4. The process according to claim 3 wherein the strain is a strainselected from the group consisting of Penicillium frequentans,Penicillium. purpurrescens, Penicillium aurantio-violaceum, Penicilliumtrzebinskii, Penicillium spinulosum, Penicillium lividum, Penicilliumjanthinellum, Penicillium turbatum,'Penicillum caseicolum, Penicilliumcrustosum, and Penicilliumt implicatumf 5. The process according toclaim 3 wherein the inorganic cation is an alkali metal cation.

6. The process according to claim 5 wherein the strain is a strain ofthe species Penicillim frequentans.

7. The process according to claim 5 wherein the strain is a strain ofthe species Penicillium aurantioviolaceum.

8. The process according to claim 5 wherein the assimilable carbonsource is a carbohydrate selected from the group consisting of dextrose,sucrose, starch, glycerol,

and molasses, and the nitrogen source is selected from the groupconsisting of cornsteep liquor, yeast extract,

soybean meal, cotton seed meal, casein, and disti'llers solubles.

9. The process according to claim 8 wherein the alkali metal cation issodium.

10. The process according to claim 8 wherein the strain is a strainselected from the group consisting of Penicillium frequentans,Penicillium purpurresc ens, Penicillium aurantio-violaceum, Penicilliumtrzebinskii, Penicilliwm spinulosum, Penicillium; lividum, Penicilliumian'thinellum, turbatum, Penicillium caseicolum, Pen icilliwm crustosum,and Penicillium implicatum.

11. The process according to claim '10 wherein the inorganic cation isan alkali metal cation.

12. The process according to claim 11 wherein the microorganism is astrain corresponding to Penicillium pm'purrescens (NRRL No. 2819).

13. The process according to claim 11 wherein the microorganism is astrain corresponding to Penicillium aurantio-violaceum NRRL N0. 762).

14. The process according to claim 11 where-in the microorganism is astrain corresponding to Penicillium frequentans (NRRL No. 1915).

References Cited by the Examiner Curtis, Plant Physiology, Vol. 32, No.1, January 1957,

pp. 56-59. Photocopy -36.

A. LOUIS MONACELL, Primary Examiner.

1. THE PROCESS FOR PRODUCING N-FORMYL HYDROXYAMINO ACETIC ACID WHICHCOMPRISES AEROBICALLY CULTIVATING A STRAIN SELECTED FROM THE GROUPCONSISTING OF PENICILLIUM FREQUENTANS, PENICILLIUM PURPURRESCENS,PENICILLIUM AURANTIO-VIOLACEUM, PENICILLIUM TRZEBINSKII, PENICILLIUMSPINULOSUM, PENICILLIUM LIVIDUM, PENICILLIUM JANTHINELLUM, PENICILLIUMTURBATUM, PENICILLIUM CASEICOLUM, PENICILLIUM CRUSTOSUM, AND PENICILLIUMIMPLICATUM IN A CULTURE MEDIUM COMPRISING ASSIMILABLE SOURCES OF CARBONAND NITROGEN UNTIL A SIGNIFICANT AMOUNT OF N-FORMYL HYDROXYAMINO ACETICACID IS PRODUCED.